CIP4 promotes lung adenocarcinoma metastasis and is associated with poor prognosis.

Aberrant epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer (NSCLC) is linked to tumor progression, metastasis and poor survival rates. Here we report the role of Cdc42-interacting protein 4 (CIP4) in the regulation of NSCLC cell invasiveness and tumor metastasis. CIP4 was highly expressed in a panel of NSCLC cell lines and normal lung epithelial cell lines. Stable knockdown (KD) of CIP4 in lung adenocarcinoma H1299 cells, expressing wild-type EGFR, led to increased EGFR levels on the cell surface and defects in sustained activation of Erk kinase in H1299 cells treated with EGF. CIP4 localized to leading edge projections in NSCLC cells, and CIP4 KD cells displayed defects in EGF-induced cell motility and invasion through extracellular matrix. This correlated with reduced expression and activity of matrix metalloproteinase-2 (MMP-2) in CIP4 KD cells compared with control. In xenograft assays, CIP4 silencing had no effect on tumor growth but resulted in significant defects in spontaneous metastases to the lungs from these subcutaneous tumors. This correlated with reduced expression of the Erk target gene Zeb1 and the Zeb1 target gene MMP-2 in CIP4 KD tumors compared with control. CIP4 also enhanced rates of metastasis to the liver and lungs in an intrasplenic experimental metastasis model. In human NSCLC tumor sections, CIP4 expression was elevated greater than or equal to twofold in 43% of adenocarcinomas and 32% of squamous carcinomas compared with adjacent normal lung tissues. Analysis of microarray data for NSCLC patients also revealed that high CIP4 transcript levels correlated with reduced overall survival. Together, these results identify CIP4 as a positive regulator of NSCLC metastasis and a potential poor prognostic biomarker in lung adenocarcinoma.

Transducer of Cdc42-dependent actin assembly promotes breast cancer invasion and metastasis.

Metastatic breast adenocarcinomas display activation signatures for signaling pathways that trigger cell motility and tissue invasion. Here, we report that the adaptor protein transducer of Cdc42-dependent actin assembly-1 (Toca-1) is expressed in highly invasive breast cancers and regulates their metastatic phenotypes. We show that Toca-1 localizes to the filamentous actin-rich core of invadopodial protrusions actively degrading the extracellular matrix (ECM). Toca-1 colocalizes with Cortactin, and we show that this interaction is mediated by the SH3 domain of Toca-1. Stable knockdown (KD) of Toca-1 expression in MDA-MB-231 cells led to a significant defect in epidermal growth factor (EGF)-induced cell migration and invasion. Toca-1 KD cells also showed significant defects in EGF- and Src-induced ECM digestion and formation of invadopodial membrane protrusions. To test the role of Toca-1 in metastasis, we achieved stable Toca-1 KD in both human and rat metastatic breast adenocarcinoma cell lines. Orthotopic tumor xenografting of control and Toca-1 KD cells in natural-killer /B-/T-cell-deficient mice revealed a significant defect in spontaneous lung metastases with Toca-1 silencing in vivo. In contrast, no defects in primary tumor growth or lung seeding following tail vein injection of Toca-1 KD cells was observed, suggesting that Toca-1 functions at an early step in the dissemination of metastatic breast tumor cells. Taken together, our results identify Toca-1 as a proinvasive protein in breast adenocarcinoma and a potential therapeutic target to limit tumor metastasis.

Characterization of a highly immunogenic Mycoplasma hyopneumoniae lipoprotein Mhp366 identified by peptide-spot array.

Enzootic pneumonia (EP) in pigs caused by Mycoplasma hyopneumoniae is a highly prevalent, chronic respiratory disease, which causes considerable economic losses in the swine industry. Most herds are vaccinated, but classical bacterin vaccines do not prevent colonization and it is not possible to detect flourishing M. hyopneumoniae infections in vaccinated herds since commonly used commercial ELISAs cannot differentiate infected from vaccinated animals. To solve this problem, new immunogenic proteins, up-regulated or solely expressed during infection, need to be identified. For this purpose a peptide-spot array was constructed which presents 105 potential linear B-cell epitopes identified by in silico analysis in 35 putative lipoproteins encoded on the genome of M. hyopneumoniae type strain 232. Subjecting this array to immunoblotting using porcine convalescent serum revealed a single strongly immunoreactive epitope on the Mhp366 protein which did not react with serum from bacterin-immunized pigs. In addition, it was not possible to detect Mhp366 in total cell lysates of in vitro grown M. hyopneumoniae strains, using a polyclonal rabbit serum raised against a recombinant GST-Mhp366 fusion protein. To investigate the possibility of using an Mhp366-based ELISA in the field for differentiating vaccinated herds with and without a flourishing infection it was shown that (i) homologues of the corresponding mhp366 gene were present in all 17 M. hyopneumoniae strains and porcine lung samples tested from different geographic origins and (ii) an ELISA based on epitope-specific synthetic peptides as solid phase antigen allowed a classification of field samples. Therefore, Mhp366 might be the first antigen identified which facilitates the detection of flourishing M. hyopneumoniae infections even in vaccinated herds.

Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase.

The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K(m) and Vmax of the fusion protein were 0.56 g/l and 51 micromol/min, respectively. The activity of purified PelAHis was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.

Use of the pre-pro part of Staphylococcus hyicus lipase as a carrier for secretion of Escherichia coli outer membrane protein A (OmpA) prevents proteolytic degradation of OmpA by cell-associated protease(s) in two different gram-positive bacteria.

Heterologous protein secretion was studied in the gram-positive bacteria Bacillus subtilis and Staphylococcus carnosus by using the Escherichia coli outer membrane protein OmpA as a model protein. The OmpA protein was found to be translocated across the plasma membrane of both microorganisms. However, the majority of the translocated OmpA was similarly degraded in B. subtilis and S. carnosus despite the fact that the latter organism does not secrete soluble exoproteases into the culture medium. The finding that purified OmpA, which was added externally to the culture medium of growing S. carnosus cells, remained intact indicates that newly synthesized and exported OmpA is degraded by one or more cell-associated proteases rather than by a soluble exoprotease. Fusion of the mature part of OmpA to the pre-pro part of a lipase from Staphylococcus hyicus allowed the efficient release of the corresponding propeptide-OmpA hybrid protein into the supernatant and completely prevented the cell-associated proteolytic degradation of the mature OmpA, most likely reflecting an important function of the propeptide during secretion of its natural mature lipase moiety. The relevance of our findings for the biotechnological use of gram-positive bacteria as host organisms for the secretory production of heterologous proteins is discussed.

Functional characterization of the Staphylococcus carnosus SecA protein in Escherichia coli and Bacillus subtilis secA mutant strains.

The Staphylococcus carnosus secA gene was cloned using the Bacillus subtilis secA gene as a probe. The S. carnosus secA encodes a polypeptide of 844 amino acid residues which is homologous to other known SecA proteins. The S. carnosus SecA functionally complemented the growth and secretion defects of a temperature-sensitive B. subtilis secA mutant at the non-permissive temperature. In contrast, the growth defect of an Escherichia coli secA mutant could not be complemented by the S. carnosus SecA protein. Our results suggest that the interactions of SecA with precursor proteins and/or other components of bacterial preprotein translocase are optimized within each organism.

A cell-free protein translocation system prepared entirely from a gram-positive organism.

A cell-free protein translocation system derived exclusively from a Gram-positive bacterium is described here for the first time. Highly efficient in vitro synthesis of plasmid encoded preprolipase of Staphylococcus hyicus is accomplished by coupled transcription/translation using either a cytosolic extract of S. carnosus alone or in combination with T7-RNA-polymerase. Addition of inside-out cytoplasmic membrane vesicles of S. carnosus leads to the partial conversion (processing) of preprolipase to prolipase. In addition, as shown in a protease protection assay, a significant part of preprolipase plus prolipase is translocated in vitro into the lumen of the vesicles. Translocation of preprolipase into the membrane vesicles requires the proton-motive force and the S. carnosus SecA protein.

The Staphylococcus carnosus secE gene: cloning, nucleotide sequence, and functional characterization in Escherichia coli secE mutant strains.

A DNA fragment containing the genes secE, nusG and rplK of Staphylococcus carnsosus was cloned using the Escherichia coli rplK gene as a probe. The S. carnosus secE homologue encodes a protein of 65 amino acid residues which is homologous to the carboxyl-terminal region of the E. coli SecE protein. The S. carnosus SecE polypeptide which, in contrast to the E. coli SecE protein, contains only one putative transmembrane segment, could fully replace the E. coli SecE protein in two different secE mutants. These results strongly suggest that the identified secE gene encodes an important component of the S. carnosus protein export apparatus.

An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtilis.

The translocation of secretory proteins derived from a Gram-positive (Staphylococcus hyicus prolipase) or a Gram-negative (Escherichia coli pre-OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. In both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre-prolipase signal peptide (38 amino acids in length) or the pre-OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre-OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram-positive and Gram-negative bacteria.

The relation between constitutional skin color and photosensitivity estimated from UV-induced erythema and pigmentation dose-response curves.

In 54 healthy volunteers we assessed predictors of sensitivity to ultraviolet (UV) light, including Fitzpatrick's sun reactive skin types and constitutional skin color, and compared these with one another and with responses of the skin to UV irradiation, as determined experimentally by a minimal erythema dose (MED), a minimal melanogenic dose (MMD), and dose-response curves for UV-induced erythema and pigmentation. For these studies, a xenon arc solar simulator was used as the source of UV irradiation, and a chromameter interfaced with a computer for objective measurement of UV-induced erythema and pigmentation was employed. The skin type did not correspond well to the constitutional skin color, as measured by a chromameter prior to UV irradiation. Within each skin type, there were large ranges of MED and MMD values and great variability in the shapes of the dose-response curves. Constitutional skin color was also not a good predictor of the measured MED and MMD values but did appear to correlate with the steepness of the dose-response curves for erythema and for pigmentation. From these studies, we propose that objectively measured constitutional skin color is a better predictor of UV responses of the skin than skin type and that steepness of dose-response curves for erythema is a better measure of the response of the skin to UV irradiation than is a MED measurement.